Investigations on plant Protease Inhibitors and their Interactions with Helicoverpa armigera mid gut Cysteine and Metalloproteases for effective control of Herbivory

Abstract

Pest control strategies against Helicoverpa armigera using protease inhibitors newlinerelied on gut protease profile of the latter larval stage of the insect which is newlinepredominated by serine proteases. Little is known about the gut protease profile newlineof the early larval instars. Specific substrates and inhibitors were used to detect newlinegut proteases active in the 3rd, 4th, 5th and late 5th instar larvae reared on artificial newlinediet. Analysis of gut protease profiles of different instars revealed differential newlineexpression of protease activity in different instars. There is also a statistically newlinesignificant variation in the specific activity of trypsin, chymotrypsin, cysteine, newlinecarboxypeptidase-A and aminopeptidase-N across instars. In general, specific newlineactivity increased from 3rd to 5th instar and then decreased at the onset of pupation newlinein late 5th instar. Optimum pH for proteolytic activity was pH 12 for the 3rd, 4th newlineand 5th instar gut extracts. newlineLevel of inhibitors against papain, carboxypeptidase-A and newlineaminopeptidase-N were estimated in 25 host plants and 25 non-host plants of H. newlinearmigera and compared. The non-host plants exhibited a statistically significant newlinehigher levels of cysteine protease inhibitors (Plt0.05) compared to the host plants. newlineA carboxypeptidase-A inhibitor and a papain inhibitor were purified from two newlinenon-host plants Oxalis corniculata and Withania somnifera with molecular newlineweights 17 ± 1.7 kDa and 18.2 ± 1.8 kDa respectively. The inhibitors are newlinethermally stable up to 75oC and are also active across a pH range from 2.6 to 10.6. newlineBoth inhibitors showed non-competitive inhibition. The carboxypeptidase-A newlineinhibitor has a Ki = 0.031 to 0.095 and#956;M and the papain inhibitor has a Ki = 0.024 newlineto 0.16 and#956;M. Both inhibitors showed high inhibition of H. armigera gut newlinecarboxypeptidase and cysteine proteases. These inhibitors were used together newlinewith Bowman-Birk inhibitor to study their individual as well as combined effects newlineon H. armigera larvae. Mortality rate was highest (40%) and larval periods newlineextended longest (39.5 ± 8.7 Days)...